CN108949749A - A method of the rapidly extracting DNA long fragment from fresh fungal mycelium - Google Patents
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Abstract
The invention discloses a kind of methods of rapidly extracting DNA long fragment from fresh fungal mycelium, prepare including thallus: weighing mycelium after taking the fungal mycelium of fresh cultured, centrifugation to filter off moisture, pulverize rapidly in liquid nitrogen；Cell cracking: DNA Extraction buffer is added, 60 μ L concentration of addition are 20mg/mL Proteinase K, in 65 DEG C of water-bath 30min after mixing；DNA is extracted: addition 0.33mL concentration is KAc, and ice bath 20min, rear phenol: chloroform: isoamyl alcohol extraction, centrifugation take supernatant that dehydrated alcohol precipitating is added, and precipitating is collected by centrifugation in stand at low temperature 30min；Purification process: RNaseA is added, in 37 DEG C of processing 30min, the phenol of rear 25:24:1: chloroform: the chloroform of isoamyl alcohol and 24:1: isoamyl alcohol is respectively extracted 1 time, low-temperature centrifugation 5min；Take supernatant that dehydrated alcohol precipitating is added, picking precipitating rinses, air-dries, be dissolved in TE buffer, save backup.2 times are improved using the filamentous fungi amount of DNA that this method is extracted, concentration reaches 277ng/ μ L, piece segment length >=20000bps, no degradation, purity is high.
Description
Technical field
The present invention relates to molecular biology fields, more particularly to one kind rapidly extracting long segment from fresh fungal mycelium
The method of DNA.
Background technique
Fungi is lower eukaryotes, enormous variety and it is various, in recent years, with the raising of high throughput sequencing technologies, more
More fungies complete gene order-checking.The in-depth analysis of genomics promotes biological evolution, phylogenetics, medicine target base
The researchs such as cause, the discovery of new gene and new edited mode and function.The length of fungal genomic DNA, concentration, purity direct relation
To the sequencing, assembling and annotation of fungal gene group.During library construction and high-flux sequence, high quality and long segment it is true
Bacterium genomic DNA can reduce stitching error, preferably reflect original gene regulating and controlling sequence and gene structure, be convenient for gene function
It is annotated with expression regulation mode.Therefore, high quality, long segment filamentous fungi extracting genome DNA scheme are quickly and easily extracted
It is particularly important in high-flux sequence, genetic engineering field.
So far, for the method for the extraction chromosome from fungi there are many research, wherein more effective method is enzyme
Solution, Benzyl chloride method, cetyl trimethylammonium bromide (CTAB) method, dodecyl sodium sulfate (SDS) method etc., latter two due to
Without expensive instrument and drug, application is more extensive.But this existing 4 kinds of common fungus genome DNA extracting methods are mainly applied
In the extraction of fungus sporophore DNA, rather than the extraction of the mycelium DNA of fresh cultured, and extraction efficiency is lower that (time is long, produces
Measure low), quality it is lower (fragment length is short, there are protein residue and catabolites).
Since fungus sporophore institutional framework is close, cell wall structure is firm, and most experiments sample is dry or many years
Raw fructification, wherein the secondary metabolites such as pigment, polysaccharide are abundant, if muscardine fructification contains 20 kinds of polysaccharide, account for about white deadlock
The total dry mass 3%-8% of bacterium；These unfavorable factors bring great difficulty to the extraction of fungal genomic DNA, lead to extraction operation
Cumbersome, the time is longer, higher cost；The yield for extracting product is lower, and degradation is serious, and the residual such as albumen, pigment is more, purity compared with
It is low.
And it is the key that extract high quality fungal genomic DNA, especially pigment, polyoses content higher that organization material is fresh
Fungi.The fungal mycelium of fresh cultured, the secondary metabolites contents such as polysaccharide, pigment are lower, and cell division proliferation is active,
DNA content is abundant, and fungal gene Engineering operation majority is using mycelium as operation object.But fresh fungal mycelium moisture content
Height causes liquid nitrogen grinding insufficient；Active enzyme content is high, leads to that DNA is degradable, long-chain is easy to be broken in operating process；In addition,
The DNA that existing in vitro fungal DNA extracting method is not suitable for fresh mycelia is extracted, and the operating time is longer (>=10h), especially
It is that lysate cracking ability is stronger, recovery rate is higher, but DNA fragmentation is short；Vice versa.Therefore do not have also a kind of from fresh fungi
The method of rapidly extracting DNA long fragment in mycelium.
Summary of the invention
In view of this, the present invention provides a kind of method of rapidly extracting DNA long fragment from fresh fungal mycelium, solution
It has determined problem of the existing technology.
The present invention provides a kind of methods of rapidly extracting DNA long fragment from fresh fungal mycelium, including following step
It is rapid:
S101 thallus prepares: mycelium 0.25g is weighed after taking the fungal mycelium of fresh cultured, centrifugation to filter off moisture,
It pulverizes rapidly in liquid nitrogen；
S102 cell cracking: being added the DNA Extraction buffer of 65 DEG C of 1mL preheatings, and 60 μ L concentration of addition are 20mg/mL egg
White enzyme K, quick oscillation mixes, in 65 DEG C of water-bath 30min；
S103DNA is extracted: addition 0.33mL concentration is 5M KAc, ice bath 20min, and the ratio that 1.5 times of volumes are added afterwards is
25:24:1 phenol: chloroform: isoamyl alcohol extraction 1 time, being centrifuged 5min at 12000rpm, take supernatant, and 2.5 times of volumes are added
Dehydrated alcohol precipitating, mixes, and precipitating is collected by centrifugation in stand at low temperature 30min at 5000rpm, several times with the rinsing of 75% ethyl alcohol,
It is rinsed 1 time with dehydrated alcohol again, drying is resuspended in 500 μ L TE buffers；
S104 purification process: 20 μ L concentration of addition are 10mg/mL RNaseA, in 37 DEG C of processing 30min, rear 800 μ L bodies
Product than be 25:24:1 phenol: chloroform: the chloroform that isoamyl alcohol and 800 μ L volume ratios are 24:1: isoamyl alcohol respectively extracts 1 time, at 4 DEG C,
5min is centrifuged under 10000rpm；Supernatant is taken, the dehydrated alcohol of 2.5 times of volumes is added, in -20 DEG C of precipitating 30min, picking is heavy
It forms sediment, is rinsed with 75% ethyl alcohol, air-dry, be dissolved in 500 μ L TE buffers, saved backup at -20 DEG C.
Preferably, in S101 step, centrifugation filters off the moisture of mycelia quality 70-75%.
Preferably, DNA Extraction buffer includes 0.1mol/L Tris-HCl, 0.5mol/L NaCl, 0.05mol/L
EDTA, 3%SDS prevent DNA degradation while rapidly extracting DNA long fragment.
Preferably, TE buffer is 10mmol/L Tris-HCl (pH 8.0), 1mmol/L EDTA (pH 8.0).
Preferably, fungi is muscardine or Cordyceps militaris.
Using a kind of method that filamentous fungi DNA is used in the sequencing of rapidly extracting long segment provided by the present invention, use is fresh
Mycelium 70%-75% dehydration, the liquid nitrogen grinding convenient for mycelia is broken and cracks；Using certain proportion Proteinase K and 3%SDS
Mixed system cracks hyphal cell and extracts DNA, not only improves lysis efficiency, saves experimental period 3-4h, influence it is fresh
High extraction is realized while retaining DNA long segment under the premise of mycelial DNA activity, in addition using RNaseA processing, phenol (pH
8.0): chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) respectively extracts 1 time, low-temperature and high-speed is centrifuged, dehydrated alcohol sinks
Shallow lake, the rinsing of 75% ethyl alcohol can both purify DNA in a mild condition, and the long segment extraction yield of 20000bp or more is more traditional
Method improves 1 times or more.
The DNA long fragment for the 20000bp or more that the method for the present invention is quickly prepared can be directly used for library construction and high pass
Sequence is measured, stitching error is reduced, preferably reflects original gene regulating and controlling sequence and gene structure, adjusted convenient for gene function and expression
Control mode annotation.
Detailed description of the invention
Fig. 1 is the muscardine mycelia schematic diagram of embodiment one；
Fig. 2 is liquid nitrogen grinding in the S101 step of embodiment one into the schematic diagram of fine-powdered；
Fig. 3 is the schematic diagram of cell pyrolysis liquid in the S102 step of embodiment one；
Fig. 4 is the schematic diagram for going out sediment after dehydrated alcohol is added in the S103 step of embodiment one；
Fig. 5 is the schematic diagram for going out flocky precipitate after dehydrated alcohol is added in the S104 step of embodiment one；
Fig. 6 extracts the electrophoresis detection of DNA solution as a result, DNA fragmentation size is essentially by embodiment one and embodiment two
20000bps, M are λ/HindIII DNA Marker, and T1 is that the method for the present invention (embodiment one and embodiment two) extracts acquisition
DNA solution, T2 are the solution that existing method (comparative example 3 and comparative example 4) obtains；
Fig. 7 is extracted the spectrophotometer testing result of DNA solution by embodiment one, wherein A260/280=1.78, A260/230
=2.32, concentration is 277ng/ μ L；
Fig. 8 is extracted the spectrophotometer testing result of DNA solution by embodiment two, wherein A260/280=1.80, A260/230
=3.11, concentration is 122ng/ μ L.
Specific embodiment
The principles and features of the present invention are described below, and illustrated embodiment is served only for explaining the present invention, is not intended to
It limits the scope of the invention.
Bacterial strain in the embodiment of the present invention is provided by Chinese Academy of Tropical Agricultural Sciences's environment and Institute of Zoology, using existing
There is method that can independently cultivate, the medicine and reagents such as Proteinase K are purchased from Reagent Company.
Embodiment one: a method of the rapidly extracting DNA long fragment from fresh muscardine mycelium, including following step
It is rapid:
S101 thallus prepares: taking on 7.5cm plate, cultivates the muscardine mycelia (such as Fig. 1) of 4d, be put into centrifuge tube
Be centrifuged 2min under 12000rpm, remove 75% moisture, after weigh mycelia 0.25g, pulverize rapidly in liquid nitrogen (as scheme
2)；
S102 cell cracking: DNA Extraction buffer (the DNA Extraction buffer: 0.1mol/L of 65 DEG C of 1mL preheatings is added
Tris-HCl (pH8.0), 0.5mol/L NaCl, 0.05mol/L EDTA, the SDS that mass concentration is 3%), 60 μ L albumen are added
Enzyme K (20mg/mL), (such as Fig. 3) quick oscillation mix, and in 65 DEG C of water-bath 30min, during which mix 3 times；
S103DNA is extracted: 0.33mL 5mol/L KAc, ice bath 20min is added, the phenol of 1.5 times of volumes is added afterwards
(0.1M Tris saturated phenol, pH > 7.5): chloroform: isoamyl alcohol (25:24:1) extracts 1 time, is centrifuged 5min at 12000rpm, takes
Supernatant is added the dehydrated alcohol precipitating of 2.5 times of volumes, mixes, stand at low temperature 30min has white flock, sheet, particle at this time
There is (such as Fig. 4) in the precipitatings such as shape, and precipitating is collected by centrifugation at 5000rpm, are rinsed 3 times with the ethyl alcohol that mass concentration is 75%, then
It is rinsed 1 time with dehydrated alcohol, drying is resuspended in 500 μ L TE buffer (10mmol/L Tris-HCl (pH 8.0), 1mmol/L
EDTA (pH 8.0)) in；
S104 purification process: 20 μ L RNaseA (10mg/mL), 37 DEG C of processing 30min, the rear phenol (pH with 800 μ L is added
8.0): chloroform: the chloroform of isoamyl alcohol (25:24:1) and 800 μ L: isoamyl alcohol (24:1) respectively extracting 1 time, at 4 DEG C, under 10000rpm
It is centrifuged 5min；Supernatant is taken, the dehydrated alcohol of 2.5 times of volumes is added, in -20 DEG C of precipitating 30min, occurs white flock precipitate at this time
(such as Fig. 5), picking precipitating, is rinsed with 75% ethyl alcohol, is air-dried, is dissolved in 500 μ L TE buffers, saves backup at -20 DEG C.
Embodiment two: a method of the rapidly extracting DNA long fragment from fresh cultured cordyceps mycelium, including it is following
Step:
S101 thallus prepare: take the Cordyceps militaris mycelia of liquid shaking table culture 4d, be put into centrifuge tube at 12000rpm from
Heart 2min removes 70% moisture, after weigh mycelia 0.25g, pulverize rapidly in liquid nitrogen；
S102 cell cracking: DNA Extraction buffer (the DNA Extraction buffer: 0.1mol/L of 65 DEG C of 1mL preheatings is added
Tris-HCl (pH8.0), 0.5mol/L NaCl, 0.05mol/L EDTA, the SDS that mass concentration is 3%), 60 μ L albumen are added
Enzyme K (20mg/mL), quick oscillation mix, and in 65 DEG C of water-bath 30min, during which mix 3 times；
S103DNA is extracted: addition 0.33mL concentration is 5mol/L KAc, ice bath 20min；The phenol of 1.5 times of volumes is added
(0.1mol/L Tris saturated phenol, pH > 7.5): chloroform: isoamyl alcohol (25:24:1) extracts 1 time, is centrifuged at 12000rpm
5min takes supernatant, and the dehydrated alcohol precipitating of 2.5 times of volumes is added, mixes, stand at low temperature 30min has white flock, piece at this time
The precipitatings such as shape, graininess occur, and precipitating is collected by centrifugation at 5000rpm, are rinsed 3 times with 75% ethyl alcohol of mass concentration, then use nothing
Water-ethanol rinses 1 time, and drying is resuspended in 500 μ L TE buffers；
S104 purification process: be added 20 μ L RNaseA (10mg/mL), 37 DEG C of processing 30min, after respectively with the phenol of 800 μ L
(pH 8.0): chloroform: the chloroform of isoamyl alcohol (25:24:1) and 800 μ L: isoamyl alcohol (24:1) respectively extracting 1 time, at 4 DEG C,
5min is centrifuged under 10000rpm；Supernatant is taken, the dehydrated alcohol of 2.5 times of volumes is added, in -20 DEG C of precipitating 30min, is occurred at this time white
Color flocculent deposit, picking precipitating, is rinsed with 75% ethyl alcohol, is air-dried, is dissolved in 500 μ L TE buffers, saves backup at -20 DEG C.
Comparative example one: the difference of comparative example one and embodiment one is, in S102 cell lysis procedure, DNA extracts buffering
SDS mass concentration is 1% in liquid, 20 μ L Proteinase Ks (20mg/ml) is added, in 65 DEG C of water-bath 45min.
Comparative example two: the difference of comparative example one and embodiment one is, in S102 cell lysis procedure, DNA extracts buffering
SDS mass concentration is 5% in liquid, and 100 μ l Proteinase Ks (20mg/ml) are added.
Comparative example three: referring to existing SDS method: taking the new fresh thalli 0.25g of muscardine, 2mL SDS is added in liquid nitrogen grinding
(0.1mol/L Tris-HCl (pH8.0), 0.5mol/L NaCl, 0.05mol/L EDTA, mass concentration are Extraction buffer
1%SDS), 65 DEG C of water-bath 1.5h；13000r/min is centrifuged 5min；Take supernatant to go in centrifuge tube, add isometric phenol/chloroform/
Isoamyl alcohol (25:24:1)；It shakes up, is centrifuged 5min, takes supernatant, add isometric chloroform/isoamyl alcohol；It shakes up, is centrifuged 3min, takes
Clearly, the dehydrated alcohol and 2mol/L NaCl of 2 times of volumes, -20 DEG C of precipitates overnights are added；12 000r/min are centrifuged 20min, go
Clearly, alcohol rinse, vacuum drying；Add 500 μ L TE, adds the RNA enzyme of 2 μ L, 37 DEG C of water-bath 1h；800 μ L chloroforms/isoamyl alcohol is added
(24:1), 12000r/min are centrifuged 10min, are repeated 2 times, natural air drying, are dissolved in 500 μ L TE buffers, save at -20 DEG C
It is spare.The difference of comparative example three and embodiment one is that new fresh thalli is without dehydration, and the concentration of SDS Extraction buffer is not
Together, not plus protease and potassium acetate processing, purification process it is different.
Comparative example four: the difference of comparative example four and comparative example three is: substituting muscardine thallus using Cordyceps militaris thallus.
DNA solution is extracted for experiment to test:
The DNA solution that DNA solution, comparative example three and comparative example four that embodiment one and embodiment two extract front and back are extracted
Electrophoresis detection result such as Fig. 6, DNA fragmentation size is essentially 20000bp, and M is λ/HindIII DNA Marker, and T1 is this hair
Bright method extracts the DNA solution obtained, and T2 is the DNA solution that existing method (comparative example 3 and comparative example 4) obtains；Embodiment one
Spectrophotometer testing result screenshot with two obtained DNA solutions is respectively such as Fig. 7 and Fig. 8.
Respectively to embodiment one to three, the DNA solution that comparative example one to four is extracted carries out electrophoresis detection and spectrophotometer
Testing result summarizes such as table 1.
1 embodiment one to three of table, the DNA solution that comparative example one to four is extracted carry out electrophoresis detection and spectrophotometer detection
As a result
As can be known from Table 1, the DNA solution that improved method of the present invention is extracted from fresh cultured fungal mycelium, genome
DNA extracted amount improves 2 times or more, and concentration reaches 277ng/ μ L, A260/280=1.8, purity is close or equal to absolute purity, card
It is bright to be polluted without albumen, phenolic substances；A260/230> 2.0,2.5 or so, illustrate in sample substantially without carbohydrate, more
The pollutants such as peptide, phenol residual；DNA fragmentation size is essentially the long segment of 20000bp, does not degrade, and is conducive to threeway and is sequenced.
In conclusion using a kind of method of rapidly extracting filamentous fungi mycelium DNA long fragment provided by the present invention,
It is dehydrated using fresh mycelia 70%-75%, the liquid nitrogen grinding convenient for mycelia is crushed and cell cracking；Using the egg centainly matched
White enzyme K and 3%SDS mixed system crack hyphal cell, not only improve lysis efficiency, but also save experimental period 3-4h, no
The DNA activity of fresh mycelia is influenced, no degradation has high extraction and pure while largely retaining DNA long segment
Degree, handled using RNaseA, phenol (pH 8.0): chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) respectively extracts 1
The centrifugation of secondary, low-temperature and high-speed, dehydrated alcohol precipitating, the rinsing of 75% ethyl alcohol can both purify DNA in a mild condition, 20000bp and
Above long segment extraction yield improves 1 times or more compared with conventional method.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent substitution, improvement and etc. done be should be included within the scope of the present invention.
Claims (5)
1. a kind of method of the rapidly extracting DNA long fragment from fresh fungal mycelium, it is characterised in that: the following steps are included:
S101 thallus prepares: mycelium 0.25g is weighed after taking the fungal mycelium of fresh cultured, rapid centrifugation to filter off moisture,
It pulverizes rapidly in liquid nitrogen；
S102 cell cracking: being added the DNA Extraction buffer of 65 DEG C of 1mL preheatings, and 60 μ L concentration of addition are 20mg/mL protease
K, quick oscillation mix, the water-bath 30min at 65 DEG C；
S103DNA is extracted: addition 0.33mL concentration is 5mol/L KAc, ice bath 20min, and the rear 0.5mL volume ratio that is added is 25:
The phenol of 24:1: chloroform: isoamyl alcohol extracts 1 time, is centrifuged 5min at 12000rpm, takes supernatant, 2.5 times of volumes are added
Dehydrated alcohol precipitating, mix, precipitating is collected by centrifugation in stand at low temperature 30min at 5000rpm, is 75% with mass concentration
Ethyl alcohol rinses several times, then is rinsed 1 time with dehydrated alcohol, and drying is resuspended in 500 μ L TE buffers；
S104 purification process: 20 μ L concentration of addition are 10mg/mL RNaseA, in 37 DEG C of processing 30min, rear 800 μ L volume ratios
For the phenol of 25:24:1: chloroform: the chloroform that isoamyl alcohol and 800 μ L volume ratios are 24:1: isoamyl alcohol respectively extracts 1 time, at 4 DEG C,
5min is centrifuged under 10000rpm；Supernatant is taken, the dehydrated alcohol of 2.5 times of volumes is added, in -20 DEG C of precipitating 30min, picking is heavy
It forms sediment, is rinsed with the ethyl alcohol that mass concentration is 75%, air-dry, be dissolved in 500 μ L TE buffers, saved backup at -20 DEG C.
2. a kind of method of rapidly extracting DNA long fragment from fresh fungal mycelium according to claim 1, feature
It is, in the S101 step, centrifugation filters off mycelia 70-75% moisture.
3. a kind of method of rapidly extracting DNA long fragment from fresh fungal mycelium according to claim 1, feature
It is, the DNA Extraction buffer includes 0.1mol/L Tris-HCl, 0.5mol/L NaCl, 0.05mol/L EDTA and 3%
SDS。
4. a kind of method of rapidly extracting DNA long fragment from fresh fungal mycelium according to claim 1, feature
It is, the TE buffer is pH of buffer=8.0 10mmol/L Tris-HCl and 1mmol/L EDTA, TE.
5. a kind of method of rapidly extracting DNA long fragment from fresh fungal mycelium according to claim 1, feature
It is, the fungi is muscardine or Cordyceps militaris.
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| CN112608918A (en) * | 2020-12-30 | 2021-04-06 | 甘肃省科学院生物研究所 | Method for quickly and simply extracting DNA of anaerobic fungi and application |
| CN113046347A (en) * | 2021-03-26 | 2021-06-29 | 山东农业大学 | Reagent and method for rapidly extracting high-quality DNA |
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| 刘少华 等: "一种快速简便的植物病原真菌基因组", 《植物病理学报》 * |
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| CN110205319A (en) * | 2019-07-18 | 2019-09-06 | 西南大学 | A kind of simple and effective reagent and method for extracting plant pathogenic fungi genomic DNA |
| CN112608918A (en) * | 2020-12-30 | 2021-04-06 | 甘肃省科学院生物研究所 | Method for quickly and simply extracting DNA of anaerobic fungi and application |
| CN113046347A (en) * | 2021-03-26 | 2021-06-29 | 山东农业大学 | Reagent and method for rapidly extracting high-quality DNA |
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Application publication date: 20181207 |